Agorins: major structural proteins of the plasma membrane skeleton of P815 tumor cells

Plasma membranes of P815 mastocytoma cells contain a set of proteins that remain selectively insoluble upon extraction of the membranes with Triton X-100, and appear to form a membrane skeletal matrix independent of the filamentous cytoskeletal systems. EGTA treatment of the matrix was found to release approximately 25% of the protein as polypeptides of 70, 69, 38, and 36 kD, all of which appear to be peripheral components associated with the cytoplasmic face of the plasma membrane via divalent cation-dependent interactions. About 75% of the total matrix protein was recovered in the EGTA-insoluble fraction. Actin accounted for approximately 5% of the total protein in the EGTA-insoluble fraction. The rest was accounted for by two novel proteins of 20 and 40 kD which, despite their relatively low molecular weights, do not enter SDS PAGE gels. Together these proteins account for approximately 15% of the total plasma membrane protein, and are thus present in much higher amounts than any other characterized protein of nucleated cell plasma membranes. Based on the extensive associations of these proteins to form very large detergent-insoluble structures, we propose that they may be named agorin I, the 20-kD protein, and agorin II, the 40-kD protein, from the Greek agora meaning assembly. The amount and properties of these proteins and the appearance of the EGTA-insoluble material in thin-section electron micrographs indicate that the agorins are the major structural elements of the membrane matrix, and thus of the putative membrane skeleton.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Role for IgE in airway secretions: IgE immune complexes are more potent inducers than antigen alone of airway inflammation in a murine model

IgE is present in airway secretions from human patients with allergic rhinitis and bronchial asthma. However, the contribution of IgE present locally to the overall airway inflammation is not well understood. We hypothesize that Ag-specific IgE can capture airborne Ags and form immune complexes. These immune complexes may function as potent inducers of immune responses in the lung, contributing to the perpetuation of airway inflammation. BALB/c mice were first sensitized with OVA in alum systemically and then challenged with nebulized OVA. Bronchoalveolar lavage (BAL) fluid from these mice contained significant amounts of IgE, of which >50% was Ag specific. The IgE levels in airway secretions remained elevated for more than 15 days after the termination of Ag exposure. Significant amounts of IgE-OVA immune complexes were detected in BAL fluid from the OVA-challenged mice. For comparison of IgE immune complexes vs Ag alone, we treated OVA-immunized mice with intranasal administration of trinitrophenyl-OVA or trinitrophenyl-OVA-anti-DNP IgE. Those treated with the immune complexes showed significantly higher levels of IL-4 and more pronounced eosinophilia in BAL fluid than did those receiving the Ag alone. The IgE immune complexes did not augment the inflammatory response in high affinity IgE receptor (FcepsilonRI)-deficient mice. We conclude that IgE present in the airways can capture the Ag and that the immune complexes thus formed may augment allergic airway response in an FcepsilonRI-dependent manner. Thus, IgE present in airway secretions may facilitate Ag-mediated allergic airway inflammation.     

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.     

Sex ratio variation shapes the ecological effects of a globally introduced freshwater fish

Sex ratio and sexual dimorphism have long been of interest in population and evolutionary ecology, but consequences for communities and ecosystems remain untested. Sex ratio could influence ecological conditions whenever sexual dimorphism is associated with ecological dimorphism in species with strong ecological interactions. We tested for ecological implications of sex ratio variation in the sexually dimorphic western mosquitofish, Gambusia affinis. This species causes strong pelagic trophic cascades and exhibits substantial variation in adult sex ratios. We found that female-biased populations induced stronger pelagic trophic cascades compared with male-biased populations, causing larger changes to key community and ecosystem responses, including zooplankton abundance, phytoplankton abundance, productivity, pH and temperature. The magnitude of such effects indicates that sex ratio is important for mediating the ecological role of mosquitofish. Because both sex ratio variation and sexual dimorphism are common features of natural populations, our findings should encourage broader consideration of the ecological significance of sex ratio variation in nature, including the relative contributions of various sexually dimorphic traits to these effects.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.     

Regeneration in the auditory system of nymphal and adult bush crickets Tettigonia viridissima

Regeneration and reestablishment of synaptic connections is an important topic in neurobiological research. In the present study, the regeneration of auditory afferents and the accompanying effects in the central nervous system are investigated in nymphs and adults of the bush cricket Tettigonia viridissima L. (Orthoptera: Tettigoniidae). In all animals in which the tympanal nerve is crushed, neuronal tracing shows a regrowth of the afferents into the prothoracic ganglion. This regeneration is seen in both adult and nymphal stages and starts 10–15 days after nerve crushing. Physiological recordings from the leg nerve indicate a recovery of tympanal fibres and a formation of functional connections to interneurones in the same time range. Electrophysiological recordings from the neck connective suggest additional contralateral sprouting of interneurones and the formation of aberrant connections. The regeneration processes of the tympanal nerve in nymphal stages and adults appear to be similar.